This proposal examines the ability of estradiol to modify effects of selective serotonin reuptake inhibitors (SSRIs), a class of drugs widely-used for the treatment of major depressive disorder (MDD). There is no clinical consensus that it is useful to add estrogen replacement therapy (ERT) to postmenopausal women with depression who are being treated with SSRIs. Our preclinical studies address this issue directly. We showed previously in 3 month old ovariectomized (OVX) rats, that estradiol (E2) administered either acutely or longer-term inhibits the SERT so as to produce antidepressant (AD)-like effects. However, E2 also blocks the inhibitory effect of SSRIs on the SERT so as to prevent their AD-like effects. Estrogen receptor ? (ER?) mediates the AD-like effects of E2 whereas ER? activation mediates its ability to block the AD effects of SSRIs. These effects were seen using the technique of chronoamperometry to measure directly SERT function in vivo. Similar results were also obtained with the forced swim test (FST), which provides a more indirect but behavioral assessment of the effects of SSRIs on the SERT. This proposal extends these observations in clinically-relevant ways by examining 1) if the age of the rats influences the effects of E2; 2) whether the length of time from ovariectomy to hormone administration impacts the effects of E2; 3) effects of E2 and age in rats expressing depressive symptoms due to exposure to chronic unpredictable stress (CUS). Although there may be no perfect animal model for the effects of estrogen deprivation in human menopause, older OVX rats are certainly more appropriate for these questions than younger ones. In Specific Aim 1 we will study the effect of age, by using OVX rats 10, 14 and 18 months old, with studies carried out 18 days post-OVX. In Specific Aim 2 we will address the critical window hypothesis of hormone action which predicts that ERT must be administered early after the menopause or after OVX to have a positive effect. We will use 10 month old OVX rats with studies carried out 4 and 8 months post-OVX. I n b o t h A i m s , in vivo chronoamperometry, carried out in anesthetized animals, will be used to measure the clearance of serotonin (5-HT) from extracellular fluid in the CA3 region of the hippocampus, which is an appropriate area of brain to study effects of both ADs and E2. ER subtype-selective agonists as well as antagonists will be used to evaluate the importance of ER subtypes in estrogen's ability to block the SERT as well as its ability to interfere with the inhibitory effect of SSRIs on the SERT. In addition, to behavioral measures will be used to examine effects of chronic hormone or drug administration: (1) the FST to evaluate AD-like effects of the hormones and SSRIs; and (2) the novelty-suppressed feeding test (NSFT) to measure their anxiolytic-like effects. Both agonist and antagonists of specific ERs will be employed to see how they alter the behavioral effects of chronic administration of hormones or SSRIs. Western blot analysis will also be used to study whether age or long term hormone depletion alters levels of ER subtypes and/or their associated signaling pathways and if long-term E2 treatment induces changes in these parameters. A final specific aim examines the effects of E2 in rats expressing depressive symptoms due to exposure to chronic unpredictable stress (CUS). These experiments will be carried out in 3 month old OVX rats to compare results to those obtained already in na?ve rats of the same age. In addition, to study effects of age on the ability of CUS to produce depressive symptomatology, these experiments will also be carried out in 10 and 18 month old OVX rats. Long term effects of E2, sertraline and subtype-selective ER agonists and antagonists will be evaluated using the FST and sucrose preference test. Our overall goal is to provide pre-clinical information relevant for the use of estrogen replacement therapy in peri- and post-menopausal depressed patients.